This project is focused on the mammary cancer preventive activity of a high-selenium garlic (abbreviated to Se-garlic). The hypothesis is that Se-garlic (and also selenium compounds contained in Se-garlic) is able to suppress the clonal expansion of mammary preneoplastic lesions by inhibiting cell proliferation and/or by stimulating apoptosis. Molecular biomarkers relevant to these two events will be studied. The rationale for supporting this hypothesis is based on new findings from animal chemoprevention bioassays and mechanistic studies in cell culture. In the rodent mammary gland, terminal end buds (TEBs) are the primary sites for the induction of mammary carcinomas by chemicals. Upon transformation, they undergo unregulated expansion to form morphologically distinct prenoeplastic lesions called "intraductal proliferations" or IDPs. Aim 1 is to determine the effect of selenium treatment on the expression of proliferative biomarkers in mammary gland TEBs and IDP lesions. These biomarkers include (a) bromodeoxyuridine labeling for DNA synthesis, (b) cyclins D1, E and A, and (c) cdk2, cdk4 and cdk6. Aim 2 is to determine the effect of selenium treatment on the expression of apoptotic biomarkers: (a) TUNEL assay by Apoptag kit, (b) apoptotic inducers bax and bad, and (c) apoptotic repressors bcl-2 and bcl-x1. All of the above biomarkers will be evaluated by immunohistochemistry. In contrast to the suppression of preneoplastic lesions, Se-garlic has minimal effect on the late stage of mammary carcinogenesis. It is well documented that preneoplastic IDPs progressively develop into more advanced neoplastic lesions called "ductal carcinoma in situ" or DCIS. The working hypothesis is that by the time the pathology has reached this stage, the DCIS lesions are no longer sensitive to selenium intervention. Aim 3 will therefore investigate the increased resistance to selenium modulation of biomarkers in these DCIS lesions. Since preliminary evidence suggested that Se-garlic has a unique effect in blocking clonal expansion of early transformed cells, it is important to find out whether this effect of Se-garlic could be mimicked by its constituent selenium compounds. Aim 4 is to examine the efficacy of methylselenocysteine and allylselenocysteine chemoprevention in the early stage of mammary carcinogenesis. Aim 5 is designed to use a gland-free fat pad transplantation approach in assessing the tumorigenic capability of preneoplastic cells from control versus Se-garlic treated rats. The objective is to corroborate the significance of clonal suppression of preneoplastic lesions in selenium protection of cancer.